Six1 is a developmental gene that is abnormally re-expressed in a large percentage of breast cancers. This over-expression plays a causal role in the initiation and metastatic development of breast cancers. Since Six1 does not have its own activation or repression domain, the Eya family of Six1 co-activator proteins is essential for Six1-mediated breast tumorigenesis and metastasis. We have developed an AlphaScreen based HTS assay targeting the Six1/Eya interaction. We propose to perform a large scale high throughput screening using the NIH MLPCN compounds to identify inhibitors of the Six1/Eya interaction. We plan to test these inhibitors for their potential as therapeutic tools to inhibit Six1-mediated breast tumorigenesis and metastasis. These inhibitors can also be used as valuable chemical probes for studies involving the Six1/Eya interaction. PUBLIC HEALTH RELEVANCE: Six1 is a transcription factor that is critical for the onset and progression of a large percentage of breast tumors, as well as for metastasis of hepatocellular carcinoma and rhabdomyosarcoma. Eya2 is a co-activator of Six1 that is essential for Six1-mediated tumorigenesis and metastasis. This project intends to identify small molecule inhibitors of the Six1/Eya interaction using high throughput screening for anti-cancer therapy. Because Six1 is overexpressed in 50% of primary breast tumors and 90% of metastatic lesions, we expect this work to potentially benefit a large population of breast cancer patients, as well as patients that harbor other types of tumors that overexpress Six1 and/or Eya.